Antigenic and catalytic disparity in the distribution of cytochrome P-450-dependent 25-hydroxyvitamin D3-1 alpha- and 24-hydroxylases.
Chick 25-hydroxyvitamin D3-1 alpha-hydroxylase, a cytochrome P-450 monooxygenase with a molecular weight of 57 kDa, can be isolated as described by Mandel et al. (1990 b). Under normal physiological circumstances, it occurs exclusively in kidney mitochondria. An isozyme of the 1 alpha-hydroxylase, known as the 24-hydroxylase, which uses the same substrate to yield an isomeric product, is also a cytochrome P-450 monooxygenase, has a molecular weight of 55 kDa, and like-wise occurs in kidney mitochondria. The amino-terminal sequences of the first 10 residues of the two isozymes are 100% homologous. Monoclonal antibodies of the IgM class raised against the 1 alpha-hydroxylase, which quantitatively discriminate against other P-450 cytochromes of mitochondrial or microsomal origin, recognize and interact with the 24-hydroxylase as an antigen. In the present study we show that the intestine, which is the only non-renal tissue with demonstrable 24-hydroxylase activity, gives a positive peroxidase-antiperoxidase immunohistochemical reaction using the monoclonal antibodies against the 1 alpha-hydroxylase. The reactions revealed that the antigen in the kidney is restricted to the cortical proximal tubular cells while in the intestine, the antigen is localized in the enterocytes of the villi. In kidney medullary or intestinal crypt cells, or in liver, heart and lung tissues where 1 alpha-hydroxylase or 24-hydroxylase activity could not be detected using cell or tissue homogenates, the immunohistochemical reactions were also negative.(ABSTRACT TRUNCATED AT 250 WORDS)
Takezawa
K
K
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Moorthy
B
B
Mandel
M L
ML
Garancis
J C
JC
Ghazarian
J G
JG
D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D000818:Animals; D002112:Calcifediol / Q000378:metabolism; D002358:Cartilage, Articular / Q000473:pathology; D002645:Chickens; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D004032:Diet; D007124:Immunoenzyme Techniques; D007413:Intestinal Mucosa / Q000201:enzymology*; D007668:Kidney / Q000201:enzymology*; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D008297:Male; D009206:Myocardium / Q000201:enzymology; D013250:Steroid Hydroxylases / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D014808:Vitamin D Deficiency / Q000201:enzymology / Q000473:pathology; D065668:Vitamin D3 24-Hydroxylase
D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D015090:25-Hydroxyvitamin D3 1-alpha-Hydroxylase; D002112:Calcifediol